Microfluidic Dialysis Protein Crystallization

Jiang HuangGN Biosystems, Inc.

March 26, 2009

Dialysis Protein Crystallization Method

Pro: scans a wide concentration range, the reagent composition can be easily altered during the course of the experiment.

Con: difficult to setup, protein consumption too high (5 to 350l/rxn), not HT compatible.

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Lysozyme

Glucoseisomerase

Catalase

Microfluidic Dialysis Plate - Design

open bottom microtiter plate

protein inlet film

dialysis membrane discs

microfluidic plate

adhesive sealing tape

protein inlet

vacuum port

dialysis membrane reagent well

Microfluidic Dialysis Plate - Design

adhesive film

Microfluidic Dialysis Plate Design

Top View

Dialysis chambers dimensions:

Screening plate:18nl per chamber (240m dia., 400m deep).

Optimization plate: 80nl per chamber (500m dia., 400m deep).

Growth plate: 1l per chamber (1.6mm dia., 400m deep)

Bottom View

Vacuum port

Dialysis chamber

Microfluidic channel

Protein port

Microfluidic Dialysis Plate – Sample Loading

FD Method Highlights

• Protein consumption as low as 15nl per dialysis

chamber

• Fast set-up in as little as 4 minutes for 96 or 384

dialysis chambers

• Low capital equipment costs to begin running

experiments

• High-throughput compatibility with standard dispensing

robotics

• Easy translation and scale-up designs

• Chemically compatible with commercial reagent kits

• The dialysis membrane can be conveniently to allow

easy loop access for crystal manipulation

FD Method Highlights

• Protein consumption as low as 15nl per dialysis

chamber

• Fast set-up in as little as 4 minutes for 96 or 384

dialysis chambers

• Low capital equipment costs to begin running

experiments

• High-throughput compatibility with standard dispensing

robotics

• Easy translation and scale-up designs

• Chemically compatible with commercial reagent kits

• The dialysis membrane can be conveniently to allow

easy loop access for crystal manipulation

FD Method Highlights

• Protein consumption as low as 15nl per dialysis

chamber

• Fast set-up in as little as 4 minutes for 96 or 384

dialysis chambers

• Low capital equipment costs to begin running

experiments

• High-throughput compatibility with standard dispensing

robotics

• Easy translation and scale-up designs

• Chemically compatible with commercial reagent kits

• The dialysis membrane can be conveniently to allow

easy loop access for crystal manipulation

Equipment and Accessories Needed

1. Vacuum pump or house vacuum with an ultimate vacuum ≤ 0.1mmHg*Air bubbles in every dialysis chamber will result due to insufficient vacuum

A List of Qualified Vacuum Pumps at under $2,000

Manufacture Model# Ultimate Vacuum

BOC/Edwards RV3 1×10-6mmHgBOC/Edwards EVA480-16-941 8×10-3mmHgWelch 1400B-01 1×10-4mmHgWelch 1399B-01 0.02mmHgBrinkmann V-500 0.01mmHgThermo-Electron 3178712 3.8×10-3mmHgThermo-Electron 3178707 1×10-4mmHg

FD Method Highlights

• Protein consumption as low as 15nl per dialysis

chamber

• Fast set-up in as little as 4 minutes for 96 or 384

dialysis chambers

• Low capital equipment costs to begin running

experiments

• High-throughput compatibility with standard dispensing

robotics

• Easy translation and scale-up designs

• Chemically compatible with commercial reagent kits

• The dialysis membrane can be conveniently to allow

easy loop access for crystal manipulation

FD Method Highlights

• Protein consumption as low as 15nl per dialysis

chamber

• Fast set-up in as little as 4 minutes for 96 or 384

dialysis chambers

• Low capital equipment costs to begin running

experiments

• High-throughput compatibility with standard dispensing

robotics

• Easy translation and scale-up designs

• Chemically compatible with commercial reagent kits

• The dialysis membrane can be conveniently to allow

easy loop access for crystal manipulation

via diameter: 1.2mm (570nl volume) via diameter: 0.3mm (20nl volume)crystal size: up to 500m long crystal size: up to 50m long

50mg/ml Lysozyme vs. HCS1 #10

Translation and Scale-up

screening optimization growthdia.=240m dia.=500m dia.=1.6mmdepth=400m depth=400m depth=400m

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Hampton Crystal Screen I

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Translation and Scale-up

FD Method Highlights

• Protein consumption as low as 15nl per dialysis

chamber

• Fast set-up in as little as 4 minutes for 96 or 384

dialysis chambers

• Low capital equipment costs to begin running

experiments

• High-throughput compatibility with standard dispensing

robotics

• Easy translation and scale-up designs

• Chemically compatible with commercial reagent kits

• The dialysis membrane can be conveniently to allow

easy loop access for crystal manipulation

FD Method Highlights

• Chemically compatible with commercial reagent kits

(materials used: PMMA, epoxy, dialysis membrane)

M. W. Toepke, D. J. Beebe, PDMS absorption of small molecules and consequences in microfluidic applications, Lab Chip, 2006, 6: 1484-1486

FD Method Highlights

• Protein consumption as low as 15nl per dialysis

chamber

• Fast set-up in as little as 4 minutes for 96 or 384

dialysis chambers

• Low capital equipment costs to begin running

experiments

• High-throughput compatibility with standard dispensing

robotics

• Easy translation and scale-up designs

• Chemically compatible with commercial reagent kits

• The dialysis membrane can be conveniently to allow

easy loop access for crystal manipulation

protein inlet

vacuum port

dialysis membrane reagent well

Microfluidic Dialysis Plate - Design

adhesive film